Anti-neoplastic, anti-viral or anti-retroviral spermine derivatives

ABSTRACT

A method of controlling the growth of mosquitos comprising administering to the mosquitos a mosquito insecticidal effective amount of a compound having one of the formulae: ##STR1## 
     
         R.sub.1 --N.sup.1 H--(CH.sub.2).sub.3 --N.sup.2 H--(CH.sub.2).sub.3 
    
      --N 3  H--(CH 2 ) 4  --N 4  H--(CH 2 ) 3  --N 5  H--(CH 2 ) 3  --N 6  H--R 6                   (II); 
     or ##STR2## wherein: R 1  and R 6  may be the same or different and are alkyl having from 1 to 12 carbon atoms or hydrocarbyl aralkyl having up to 12 carbon atoms; 
     R 2  -R 5  may be the same or different and are H, R 1  or R 6  ; 
     R 7  is H, alkyl having from 1 to 12 carbon atoms, hydrocarbyl aryl or hydrocarbyl aralkyl, each having up to 12 carbon atoms; 
     m is an integer from 3 to 6, inclusive; 
     n is an integer from 3 to 6, inclusive; or (IV) a salt thereof with an acid and a carrier therefor.

BACKGROUND OF THE INVENTION

This invention was made with U.S. Government support under GrantNCDDG-CA37606, awarded by the National Cancer Institute. The U.S.Government has certain rights in this invention.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/481,864 filed Jun. 7,1995, (now abandoned), which is a division of application Ser. No.08/162,776 filed Dec. 8, 1993 (now U.S. Pat. No. 5,455,277 issued Oct.3, 1995), which is a division of application Ser. No. 07/834,345 filedFeb. 12, 1992 (now U.S. Pat. No. 5,342,945 issued Aug. 30, 1994) whichis a division of application Ser. No. 07/210,520 filed Jun. 23, 1988(now U.S. Pat. No. 5,091,576 issued Feb. 25, 1992) which is acontinuation-in-part of application Ser. No. 07/066,227 filed Jun. 25,1987 (now abandoned), which is a continuation-in-part of applicationSer. No. 06/936,835 filed Dec. 2, 1986 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to anti-neoplastic and anti-psoriasispharmaceutical compositions and methods of treatment and to insecticidalcompositions and methods of controlling the growth of insects.

In recent years a great deal of attention has been focused on thepolyamines, e.g., spermidine, norspermidine, homospermidine,1,4-diaminobutane (putrescine), and spermine. These studies have beendirected largely at the biological properties of the polyamines probablybecause of the role they play in proliferative processes. It was shownearly on that the polyamine levels in dividing cells,e.g., cancer cells,are much higher than in resting cells. See Janne et al, A. Biochim.Biophys. Acta. 473, 241 (1978); Fillingame et al, Proc. Natl. Acad. Sci.U.S.A. 72:4042 (1975); Metcalf et al, J. Am. Chem. Soc. 100:2551 (1978);Flink et al, Nature (London) 253:62 (1975); and Pegg et al, PolyamineMetabolism and Function, Am. J. Cell. Physiol. 243:212-221 (1982).

Several lines of evidence indicate that polyamines, particularlyspermidine, are required for cell proliferation: (i) they are found ingreater amounts in growing than in non-growing tissues; (ii) prokaryoticand eukaryotic mutants deficient in polyamine biosynthesis areauxotrophic for polyamines; and (iii) inhibitors specific for polyaminebiosynthesis also inhibit cell growth. Despite this evidence, theprecise biological role of polyamines in cell proliferation isuncertain. It has been suggested that polyamines, by virtue of theircharged nature under physiological conditions and their conformationalflexibility, might serve to stabilize macromolecules such as nucleicacids by anion neutralization. See Dkystra et al, Science, 149:48(1965); Russell et al, Polyamines as Biochemical Markers of Normal andMalignant Growth (Raven, N.Y., 1978); Hirschfield et al, J. Bacteriol.,101:725 (1970); Morris et al, ibid, p. 731; Whitney et al, ibid, 134:214(1978); Hafner et al, J. Biol. Chem., 254:12419 (1979); Cohn et al, J.Bacteriol. 134:208 (1978); Pohjatipelto et al, Nature (London), 293:475(1981); Mamont et al, Biochem. Biophys. Res. Commun. 81:58 (1978);Bloomfield et al, Polyamines in Biology and Medicine (D. R. Morris andL. J.. Morton, Eds.--Dekker, N.Y., 1981) pp. 183-205; Gosule et al,Nature, 259:333 (1976); Gabbay et al, Ann. N.Y. Acad. Sci., 171:810(1970); Suwalsky et al, J. Mol. Biol., 42:363 (1969) and Liquori et al,J. Mol. Biol., 24:113 (1968).

However, regardless of the reason for increased polyamine levels thephenomenon can be and has been exploited in chemotherapy. See Sjoerdsmaet al, Butterworths Int. Med. Rev.: Clin. Pharmacol. Ther. 35:287(1984); Israel et al, J. Med. Chem., 6:1 (1973); Morris et al,Polyamines in Biology and Medicine; Dekker, N.Y., p. 223 (1981) and Wanget al, Biochem. Biophys. Res. Commun., 94:85 (1980).

It is an object of the present invention to provide novelanti-neoplastic, -viral and -retroviral compounds, pharmaceuticalcompositions and methods of treatment.

SUMMARY OF THE INVENTION

The foregoing and other objects are realized by the present invention,one embodiment of which is a pharmaceutical composition comprising ananti-neoplastic, anti-viral, anti-retroviral or anti-psoriasis effectiveamount of a compound, having one of the formulae: ##STR3##

    R.sub.1 --N.sup.1 H--(CH.sub.2).sub.3 --N.sup.2 H--(CH.sub.2).sub.3 --N.sup.3 H--(CH.sub.2).sub.4 --N.sup.4 H--(CH.sub.2).sub.3 --N.sup.5 H--(CH.sub.2).sub.3 --N.sup.6 H--R.sub.6                  (II);

or ##STR4##

Wherein:

R₁ and R₆ may be the same or different and are H, alkyl or aralkylhaving from 1 to 12 carbon atoms,

R₂ -R₅ may be the same or different and are H, R₁ or R₆ ;

R₇ is H, alkyl, aryl or aralkyl having from 1 to 12 carbon atoms

m is an integer from 3 to 6, inclusive,

n is an integer from 3 to 6, inclusive; and

a pharmaceutically acceptable carrier therefor.

An additional embodiment of the invention comprises a method of treatinga human or non-human animal in need of anti-neoplastic, anti-viral,anti-retroviral or anti-psoriasis therapy comprising administering tothe animal an anti-neoplastic, anti-viral, anti-retroviral oranti-psoriasis effective amount of a compound having one of the aboveformulae.

A further embodiment of the invention comprises a compound having theformula: ##STR5##

Wherein:

R₁ -R₆ may be the same or different and are methyl, propyl, butyl,pentyl, benzyl or β,β,β-trifluoroethyl;

m is an integer from 3 to 6, inclusive;

n is an integer from 3 to 6, inclusive.

A further embodiment of the invention comprises a compound having theformula:

    R.sub.1 --N.sup.1 H--(CH.sub.2).sub.3 --N.sup.2 H--(CH.sub.2).sub.3 --N.sup.3 H--(CH.sub.2).sub.4 --N.sup.4 H--(CH.sub.2).sub.3 --N.sup.5 H--(CH.sub.2).sub.3 --N.sup.6 H--R.sub.6                  (II);

Wherein: R₁ and R₆ may be the same or different and are alkyl or aralkyl

having from 1 to 12 carbon atoms.

A final embodiment of the invention comprises a compound having theformula: ##STR6##

Wherein:

R₁ and R₆ may be the same or different and are alkyl or aralkyl havingfrom 1 to 2 carbon atoms;

R₇ is H, alkyl, aralkyl or aryl having from 1 to 12 carbon atoms;

n is an integer from 3 to 6, inclusive.

DETAILED DESCRIPTION OF THE INVENTION

In compounds of the invention, R₁ and R₆ are preferably methyl, ethyl,propyl, benzyl, etc., it being understood that the term "aralkyl" isintended to embrace any aromatic group the chemical and physicalproperties of which do not adversely affect the efficacy and safety ofthe compound for therapeutic applications. Preferred, however, are thehydrocarbyl aralkyl groups, i.e., comprised only of C and H atoms.

R₂ -R₅ preferably are H, methyl, ethyl, propyl or benzyl.

Compounds of formula (I) are preferably synthesized by first forming asulfonamide of the polyamine at all of the amino nitrogens (1) toactivate the primary amines for monoalkylation, and (2) to protect anysecondary nitrogens from alkylation. Suitable sulfonating agents includealkyl, aryl and arylalkyl sulfonating agents of the general structureRSO₂ X wherein R is alkyl, aryl or arylalkyl and X is a leaving group,e.g., Cl⁻, Br⁻, etc. The sulfonation is accomplished by reacting thepolyamine with 1.0 equivalent of sulfonating agent per nitrogen in thepresence of a base, e..g., tertiary amine or a hydroxide. The reactionis best accomplished using aqueous sodium hydroxide as the base andp-toluenesulfonyl chloride (TsCl) as the sulfonating agent in a bipbasicsolvent system consisting of an organic solvent, e.g., methylenechloride and water. The sulfonating agent is added in methylene chlorideto an aqueous solution of the amine and sodium hydroxide and thereaction proceeds according to the following equation, using spermine asthe base compound: ##STR7##

Wherein:

Ts=p-toluenesulfonyl.

After purification the sulfonamide is next alkylated. The alkylationsinvolve formation of N-anions on the primary amino sulfonamides with abase such as NaH followed by reaction of the N-anion with an alkylatingagent RX wherein R is as defined above and X is a leaving group such asI⁻, CI⁻, Br⁻, p-CH₃ C₆ H₄ SO₃ ⁻, CH₃ SO₃ ⁻.

The alkylation can be carried out in a variety of dipolar aproticsolvents, preferably, N, N-dimethyl-formamide (DMF), The reactionproceeds according to the following equation: ##STR8##

After alkylation of the sulfonamide, the sulfonyl protecting groups arenext removed under reducing conditions. Although a variety of standardreducing conditions can be utilized (LiAlH₄, Li/NH₃, catalyticreduction), Na and NH₃ function optimally. The reduction proceedsaccording to the following equation: ##STR9##

The compounds are isolated as the free amines and then may be convertedto and utilized as the corresponding hydrochloride salts by treatmentwith concentrated HCl. However, they may also be used as salts with anypharmaceutically acceptable acid, e.g., HBr, CH₃ CO₂ H, CH₃ SO₃ H, etc.

Compounds of formula (II) are preferably prepared by the mono-alkylationof tetratosyl spermine at each of the primary nitrogens by reagents suchas N-alkyl-N-(3-chloropropyl)-p-toluenesulfonamide. Terminal alkylationof spermine is carried out using the conditions employed for preparingcompound (I) according to the following scheme: ##STR10##

The alkylating agent is formed by treatment ofN-alkyl-p-toluenesulfonamide with excess 1,3dichloro-propane under theaforementioned conditions according to the following scheme: ##STR11##

After purification of the dialkylated hexatosylated hexaamine, thesulfonyl protecting groups are removed reductively with sodium in liquidammonia and THF as follows: ##STR12## The final product is isolated asthe free amine and may be converted to the hydrochloride salt.

Compounds of formula (III) may be prepared by reacting a tetraamine offormula (I) in which R₂ -R₅ ═H and R₁, R₆ =alkyl or aralkyl with twoequivalents of an aldehyde R₇ CHO, wherein R₇ ═H, alkyl or aralkyl.

Specifically, to N¹,N⁴ -diethylspermine tetrahydrochloride is addedaqueous NaOH and formalin (two equivalents) to generate thebis-hexahydropyrimidine as follows: ##STR13##

The invention is illustrated by the following non-limiting examples.

EXAMPLE 1 Preparation of N¹,N⁴ -diethylspermine N¹,N²,N³,N⁴-Tetra-p-tosylspermine. To spermine tetrahydrochloride (4.53 g, 13.0mmol) and 10% aqueous NaOH (200 mL, 132 mmol) at 0° is added dropwisep-toluene-sulfonyl chloride (9.98 g, 52.3 mmol) in CH₂ Cl₂ with rapidstirring. After 1 hr the mixture is allowed to warm to room temperatureand to stir for 2 days. The organic phase is separated and washed with0.5N HCl, H₂ O, and brine, dried over Na₂ SO₄ and purified on silica gel(450 g, 3% MeOH/CHCl₃) to give 9.69 g, 91% yield of tetratosylspermine.

NMR (CDCl₃) δ 7.2-7.9 (m, 16H), 5.34 (t, 2H, J=7), 2.9-3.3 (m, 12H),2.43 (s, 12H), 1.5-2.0 (m, 8H).

N¹,N⁴ -Diethyl-N¹,N²,N³,N⁴ -Tetra-p-tosyl-spermine. To thetetratosylspermine prepared above (1.75 g, 2.14 mmol) in dry DMF (12 mL)was cautiously added 80% sodium hydride (0.25 g, 8.33 mmol) and thenethyl iodide (1.0 mL, 12.5 mmol). After heating under nitrogen (10 h,55°), the mixture was quenched with ice water and extracted withchloroform (3×). The organic phase was washed with 5% Na₂ SO₃, 5% NaOH,1N HCl, and water, then dried with Na₂ SO₄. Removal of DMF by flashdistillation and purification of the crude product on silica gel (4%EtOH/CHCl₃) produced 1.63 g (87%) of the desired product. NMR (CDCl₃) δ7.2-7.8 (m, 16H), 3.03-3.3 (m, 16H), 2.43 (s, 12H),1.5-2.1 (m, 8H), 1.08(t, 6H, J=7). Anal. Calcd. for C₂₄ H₅₈ N₄ O₈ S₄ ; C, 57.64; H, 6.68; N,6.40. Found: C, 57.69; H, 6.74; N, 6.20.

N¹,N⁴ -diethylspermine (DES). Into a solution of the N¹,N⁴-diethyl-N¹,N²,N³,N⁴ -tetratosyl-spermine prepared above (2.78 g, 3.18mmoles) in dry, distilled THF (200 mL) at -78° C. was condensed 300 mLNH₃, using a dry ice condenser. Sodium spheres (3.0 g, 0.13 mol) werethen added in small portions and the reaction mixture was stirred at-78° C. for 4 h. The reaction mixture was allowed to warm to roomtemperature overnight and the NH₃ boiled off. Diethyl ether was added tothe mixture. Ethanol was then cautiously added, then H₂ O was added tofinally quench the reaction. The solvents were evaporated and theproduct extracted with diethyl ether and then chloroform. The extractswere dried over Na₂ SO₄, filtered and the extracts concentrated. Theresultant liquid was distilled in a Kugelrohr apparatus (150° C., 0.1mm). Concentrated hydrochloric acid was added to an ether/ethanol (1:1)solution of the distillate to form the hydrochloride salt, which wasrecrystallized from hot aqueous ethanol to give 790 mg (63%) DES. NMR(D₂ O) δ 1.4 (t, 6H); 1.9 (m, 4H); 2.25 (m, 4H); 3.25 (m, 16H); 4.80 (s,HOD, reference).

The following protocols were followed to determine the IC₅₀ values forDES against cultured L1210 cells, Daudi cells and HL-60 cells.

Cell Culture.

Murine L1210 leukemia cells, human Burkitt lymphoma cells (Daudi) andhuman promyelocytic leukemia cells (HL-60) were maintained inlogarithmic growth as suspension cultures in RPMI-1640 medium containing2% 4-(1-hydroxyethyl)-1-piperazineethanesulfonicacid/3-(N-morpholino)propanesulfonic acid, 100 μM aminoguanidine, and10% fetal bovine serum. Cells were grown in 25 sq cm tissue cultureflasks in a total volume of 10 mL under a humidified 5% CO₂ atmosphereat 37° C.

The cells were treated while in logarithmic growth (L1210 cells 0.3×10⁵cells/mL; Daudi and HL-60 1×10⁵ cells/mL) with the polyamine derivativesdiluted in sterile water and filtered through a 0.2 micron filterimmediately prior to use. Following a 48 h incubation with L1210 cellsand a 72 h incubation with Daudi or HL-60 cells, L1210 cells werereseeded at 0.3×10⁵ cells/mL, Daudi and HL-60 cells were reseeded at1×10⁵ cells/mL and all cells were incubated in the presence of thepolyamine derivative for an additional 48 h or 72 h.

Cell samples at the indicated time periods were removed for counting.Cell number was determined by electronic particle counting and confirmedperiodically with hemocytometer measurements. Cell viability wasassessed by trypan blue dye exclusion.

The percentage of control growth was determined as follows: ##EQU1## TheIC₅₀ is defined as the concentration of compound necessary to reducecell growth to 50% of control growth.

The results are set forth in Tables 1 and 2.

                  TABLE 1                                                         ______________________________________                                        L1210 Cells                                                                                 48 H IC.sub.50                                                                         96 H IC.sub.50                                         ______________________________________                                        DES           10 μM 0.10 μM                                             ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Daudi Cells             HL-60 Cells                                                  72 H IC.sub.50                                                                         144 H IC.sub.50                                                                           72 H IC.sub.50                                                                       144 H IC.sub.50                            ______________________________________                                        DES    >40 μM                                                                              0.5 μM   10 μM                                                                             0.3 μM                                  ______________________________________                                    

Animal Studies.

The murine L1210 leukemia cells were maintained in DBA/2J mice. L1210cells, from a single mouse which was injected i.p. with 10⁶ cells 5 daysearlier, were harvested and diluted with cold saline so that there were10⁵ or 10⁶ cells in 0.25 cc. For each study, mice were injected i.p.with 10⁶ L1210 cells or 10⁵ L1210 cells (See Table 3) on day 0. Thepolyamine analogues were diluted in sterile saline within 24 h of useand the unused portion stored at 5° C.

DES was administered by i.p. injection 15 mg/kg or 20 mg/kg every 8 hfor 3 days (days 1-3), 4 days (days 1-4), or 6 days (days 1-6) (seeTable 3).

Mice which were treated with saline injections served as controls.

The parameter used for treatment evaluation was mean survival time.

(Percent increased life span, % ILS). ##EQU2##

The murine Lewis lung carcinoma was maintained as s.c. tumor in C57B1/6mice. The line was propagated every 4 days. A 2-4 mm fragment of s.c.donor tumor was implanted s.c. in the axillary region with a puncture inthe inguinal region on day 0.

DES was administered by i.p. injection 20 mg/kg every 8 h for 5 daysbeginning on day 5 (days 5-9). Equal numbers of mice treated with salineinjections served as controls. The parameter used for treatmentevaluation was mean survival time (% ILS).

The parameters of the animal tests and results are set forth below inTables 3 and 4.

                                      TABLE 3                                     __________________________________________________________________________    Evaluation of DES in DBA/2J                                                   Male Mice with L1210 Leukemia (i.p.)                                          DES                                                                              Dosing Schedule                                                                       No. Animals                                                                         Day of Death                                                                              Mean Survival SD                                                                       % ILS                                   __________________________________________________________________________    1).sup.a                                                                         15 mg/kg q12 hr                                                                       6     14, 14, 14.5, 15,                                                                         14.9 ± 1.3                                                                          55                                         days 1-6      15, 17                                                          Control 7     8.5, 9.5, 9.5, 9.5,                                                                       9.6 ± 0.5                                                                           0                                                        10, 10, 10                                                   2).sup.b                                                                         20 mg/kg q8 hr                                                                        4     13.5, 14, 14, 14.5                                                                        14.1 ± 0.5                                                                          57                                         days 1-3                                                                      Control 4     8.5, 8.5, 9, 10                                                                           9.0 ± 0.5                                                                           0                                       3).sup.b                                                                         20 mg/kg g8 hr                                                                        10    14, 14, 15, 15, 16,                                                                       16.7 ± 2.6                                                                          90                                         days 1-4      17, 18, 20, 21, 31                                              Control 9     8, 8, 9, 9, 9, 9, 9,                                                                      8.8 ± 0.4                                                                           0                                                        9, 10                                                        4).sup.a                                                                         15 mg/kg q8 hr                                                                        8     8, 20, 22.5, 24.5,                                                                        27.8 ± 19.5                                                                         302                                        days 1-6      28.5, 60.sup.d, 60.sup.d, 60.sup.d                              Control 6     8.5, 9.5, 10, 10, 10.5, 10.5                                                              9.8 ± 0.7                                                                           0                                       __________________________________________________________________________     .sup.a Mice injected with 10.sup.5 L1210 cells i.p. on day 0.                 .sup.b Mice injected with 10.sup.6 L1210 cells i.p. on day 0.                 .sup.c Death of animal not included in statistics . . . greater or less       than Mean Survival 2x (S.D).                                                  .sup.d Experiment ended at 60 days. Animal survival evaluated on this day     however, these animals were alive with no sign of tumor.                 

                  TABLE 4                                                         ______________________________________                                        Evaluation of N.sup.1, N.sup.4 -Di-ethylspermine (DES) in                     C57B1/6J Male Mice with Lewis Lung Carcinoma (s.c.)                                   Dose             Survival Values (Days)                               Drug      (mg/kg)  Schedule  Mean ± S.D.                                                                         % ILS                                   ______________________________________                                        DES       20 (i.p.)                                                                              Every 8 h,                                                                              43.7 ± 7.1                                                                          24                                                         days 5-9                                                   Control (Saline)                                                                        --       --        35.2 ± 2.6                                                                           0                                      ______________________________________                                    

The foregoing test results unequivocally establish the effectiveness ofthe composition of the invention as an anti-neoplastic agent.

EXAMPLE 2

N-Ethyl-N-(3-chloropropyl)-p-toluenesulfonamide. ToN-ethyl-p-toluenesulfonamide (5.01 g, 0.0251 mol) in DMF (50 mL) in adry flask is added sodium hydride (80% in oil, 0.93 g, 0.031 mol). Aftergas evolution subsides, 1,3-dichloropropane (22.48 g, 0.199 mol) isadded. The mixture is heated at 53° C. for 10 h then cooled and pouredinto ice water (300 mL), which is extracted twice with ether. Thecombined extracts are washed with 1% sodium bisulfite, water (3x), andbrine. Removal of solvent by rotary evaporation then Kugelrohrdistillation gives crude product, which is chromatographed on silica gel(30% hexane/CHCl₃) to furnish 2.91 g product (42%) NMR (CDCl₃) δ 1.15(t, 3H), 1.9-2.2 (m, 2H), 2.44 (s, 3H), 3.11-3.35 (m, 4H), 3.6 (t, 2H),7.3 (d, 2H), 7.74 (d, 2H).

3,7,11,16,20,24-Hexa(p-toluenesulfonyl)3,7,11,16,20,24-hexaazahexacosane.To tetra(p-toluenesulfonyl) spermine (1.82 g, 2.22 mmol) in dry DMF (10mL) is added sodium hydride (80% in oil, 0.21 g, 7.0 mmol) and potassiumiodide (53 mg, 0.32 mmol). After 30 minutes,N-ethyl-N-(3-chloropropyl)-p-toluenesulfonamide (2.9 g, 10.5 mmol) inDMF (10 mL) is introduced and the mixture is stirred for 20 h at roomtemperature then heated at 40°-50° C. for 2 h. The cooled reactionmixture is poured into ice-cold 5% NaOH (100 mL), which is extractedwith CHCl₃ (3×). A water wash, then solvent removal (rotovap thenKugelrohr distillation) yields crude hexatosylamide. Silica gelchromatography (1% EtOH/CHCl₃) affords 1.73 g of product (60%). NMR δ1.08 (t, 6H), 1.45-2.10 (m, 12H), 2.34 (s, 18H), 2.96-3.37 (m, 24H),7.2-7.8 (m, 24H).

1,20-Bis(N-ethylamino)-4,8,13,17-tetraazaeicosane. A solution of thepreceding compound (0.79 g, 0.61 mmol) in distilled THF (45 mL) is addedto a dry 500 mL, 3-necked flask, equipped with a dry ice condenser and 2stoppers. The solution is cooled to about -40° C., and ammonia gas (200mL), after passing through NaOH, is condensed. Sodium spheres (0.99, 43mmol), which are rinsed in hexane (2×) and cut in half, are addedcautiously. After maintaining the cold temperature for 4-5 h, ammoniagas is allowed to evaporate under a stream of nitrogen. To the residueat 0° C. is carefully added excess, absolute ethanol, and the mixture isconcentrated. Sodium hydroxide (10%, 15 mL) is then added, andextraction with chloroform (10×20 mL), while saturating the aqueouslayer with salt, gives crude free amine. Bulb-to-bulb distillation, upto 160° C./0.005 mm, furnishes 0.216 g free hexaamine, which isdissolved in ethanol and treated with 0.5 mL concentrated HCl. Aftersolvent removal, the solid is recrystallized from 17% aqueous ethanol(120 mL) and washed with cold, absolute EtOH (2×3 mL) to afford 0.131 gof crystalline product (35%). 300 MHz NMR (D₂ O) δ 1.3 (t, 6H),1.74-1.84 (m, 4H), 2.05-2.19 (m, 8H), 3.07-3.25 (m, 24H). Anal. calcd.for C₂₀ H₅₄ Cl₆ N₆ : C, 40.62; H, 9.20; N 14.21. Found: C, 40.73; H,9.22; N, 14.22.

EXAMPLE 3

Bis(3-ethyl-1-hexahydropyrimidyl)-1,4-butane. To N¹, N⁴-diethylspermine.4HCl (36.1 mg, 0.0893 mmol) in 0.17M NaOH (2.0 mL, 0.34mmol) at 0° is added formalin (15 μL, 0.20 mmol). The solution isstirred at room temperature for 3 h, then 10% NaOH (4 mL) and brine (4mL) are added. Extraction with CH₂ Cl₂ (4×25 mL) and drying the extractswith Na₂ SO₄ gives crude product. Column chromatography (silica gel, 2%concentrated NH₄ OH/CH₃ OH) furnishes 22 mg (88% yield) of thebis-hexahydropyrimidine. NMR (CDCl₃) δ 1.10 (t, 6H), 1.4-1.9 (m, 8H),2.32-2.65 (m, 16H), 3.15 (s, 4H).

EXAMPLE 4

The IC₅₀ values for several compounds according to the invention weredetermined as in Example 1 and 2. The results are set forth in Table 5.

                  TABLE 5                                                         ______________________________________                                        L-1210 Cells  IC.sub.50 !                                                     Compound             48 hrs.   96 hrs.                                        ______________________________________                                        Formula I -                                                                            R.sub.1 = R.sub.6 = methyl                                                                    60%    CG   0.75 μM                                        m = 3           100    μM                                                  n = 4                                                                         R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H                            Formula I -                                                                            R.sub.1 = R.sub.6 = propyl                                                                    3      μM                                                                              0.2  μM                                        m = 3                                                                         n = 4                                                                         R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H                            Formula I -                                                                            R.sub.1 = R.sub.2 = R.sub.5 = R.sub.6 = ethyl                                                 80%    CG   5    μM                                        R.sub.3 = R.sub.4 = 11                                                                        25     μM                                                  m = 3                                                                         n = 4                                                                Formula I -                                                                            R.sub.1 = R.sub.3 = R.sub.4 = R.sub.6 = ethyl                                                 100    μM                                                                              3    μM                                        R.sub.2 = R.sub.5 = 11                                                        m = 3                                                                         n = 4                                                                Formula II -                                                                           R.sub.1 = R.sub.6 = ethyl                                                                     50     μM                                                                              0.5  μM                               ______________________________________                                    

EXAMPLE 5

The % ILS value for various dosages of N¹,N⁴ -diethylhomospermine weredetermined according to the procedure of Examples 1 and 2. The resultsare set forth in Table 6.

                  TABLE 6                                                         ______________________________________                                        L1210 i.p. Leukemia in DBA/2J female mice 10.sup.5 cells on day 0.                                      Day of                                                                              Mean Survival +                                                                         ILS                                 No.  Dosing Schedule                                                                           # Animals                                                                              Death S.D. (days)                                                                             (%)                                 ______________________________________                                        1.   2.5 mg/kg q 8 hr                                                                          5        20.5, 32                                                                            31.5 ± 16.6                                                                          242                                      days 1-6 (i.p.)      23, 22,                                                                       60.sup.a                                                 Control                    9.2 ± 0.3                                  2.   5 mg/kg q 8 hr                                                                            10       25,   56.5 ± 11.1                                                                          524                                      days 1-6 (i.p.)      9 × 60.sup.a                                       Control                    9.1 ± 0.6                                  3.   10 mg/kg q 12 hr                                                                          6        31,   55.2 ± 11.8                                                                          441                                      days 1-6 (i.p.)      5 × 60.sup.a                                       Control                    10.2 ± 1.1                                 4.   10 mg/kg once                                                                             5        12, 17,                                                                             20.0 ± 6.1                                                                           115                                      daily days (1-6      24, 24,                                                  (i.p.)               27                                                       Control                    9.3 ± 0.4                                  5.   15 mg/kg once                                                                             5        21, 27,                                                                             45.6 ± 19.8                                                                          390                                      daily days (i.p.)    3 × 60.sup.a                                       Control                    9.3 ± 0.3                                  ______________________________________                                         .sup.a Experiment ended at 60 days. Animal survival evaluated on this day     however, these animals were alive with no sign of tumor.                 

Unexpectedly, and for reasons as not yet understood, the compounds ofthe invention have been found to be effective anti-viral, and mostsurprisingly, anti-retroviral agents.

The development of compounds useful for the prophylaxis and therapy ofviral disease has presented more difficult problems than thoseencountered in the search for drugs effective in disorders produced byother microorganisms. This is primarily because, in contrast to mostother infectious agents, viruses are obligate intracellular parasitesthat require the active participation of the metabolic processes of theinvaded cell. Thus, agents that may inhibit or cause the death ofviruses are also very likely to injure the host cells that harbor them.Although the search for substances that might be of use in themanagement of viral infections has been long and intensive, very fewagents have been found to have clinical applicability. Indeed, eventhese have exhibited very narrow activity, limited to one or only a fewspecific viruses.

The retroviruses have presented an even greater challenge due to theireven more complex intracellular metabolic activity.

The following examples illustrate the utilization of the compounds ofthe present invention as anti-retrovirus agents.

EXAMPLE 6

Embryonic chicken fibroblasts were grown to near confluence in cellculture media. The fibroblasts were next exposed to arian sarcoma virusfor five hours. The cells were next washed with buffer to remove excessvirus. The virus infected cells were then treated with 100 μM or 100 μM,N¹,N⁴ -diethylspermine, (DES), in culture media for 18 hours. The cellculture media was next removed and the cells were overlaid with softagar growth media. The cells were then allowed to grow at 37° C. for 6-8days. The culture plates were evaluated for foci (transformed cells)utilizing an inverted microscope. The results of these measurements areindicated below.

                  TABLE 7                                                         ______________________________________                                        NUMBER OF FOCI AT    6-DAYS   8-DAYS                                          ______________________________________                                        CONTROL (ASV + FIBROBLASTS)                                                                        300      300                                             ASV + FIBROBLASTS + 10 μM DES                                                                    20      300                                             ASV + FIBROBLASTS + 100 μM DES                                                                   0       110                                             ______________________________________                                    

In a second experiment the virus was first treated with DES at 10 μM or100 μM for three hours and then added to the fibroblast monolayer for 18hours at 37° C. The excess virus was then removed by washing and themonolayer overlaid with soft agar culture media. The plates were allowedto incubate at 37° C. for 8 days and the plates were examined for foci.The results are indicated as follows.

                  TABLE 8                                                         ______________________________________                                                        NUMBER OF FOCI AT 8 DAYS                                      ______________________________________                                        ASV + FIBROBLASTS 300                                                         (CONTROL)                                                                     ASV + FIBROBLASTS + 10 μM                                                                    200                                                         DES                                                                           ASV + FIBROBLASTS + 100 μM                                                                    16                                                         DES                                                                           ______________________________________                                    

Inasmuch as the compounds described herein are anti-proliferationagents, they are also useful as anti-psoriasis agents. The followingexample illustrates the transdermal penetration characteristics of thecompounds of the invention.

EXAMPLE 7

Hairless mice were sacrificed by cervical dislocation and their skinremoved. The skin was denuded of fatty tissue and stretched over a drugdiffusion cell. The diffusion cell contained a phosphate receptor phaseat pH 7.4. The donor phase contained the drug DES dissolved in glycinebuffer at pH 8.0 at a concentration of 10 mg/mL. Samples of the receptorphase (3 mL) were taken at 48 hours. After each sample was withdrawn, anequal volume of fresh receptor phase was added back. The samples removedfrom the diffusion cell were assayed for polyamine utilizing a liquidchromatography-C-18 reverse system. The samples were first acidifiedwith perchloric acid and then reacted with dansyl chloride to producethe corresponding dansylated polyamines. The experiment revealed thatDES did indeed cross the skin at the dermal barrier.

For each of the utilities mentioned herein, the amount required ofactive agent and the frequency of its administration will vary with theidentity of the agent concerned and with the nature and severity of thecondition being treated and is of course ultimately at the discretion ofthe physician or veterinarian. In general, however, a suitable dose ofagent will lie in the range of about 1 mg to about 200 mg per kilogrammammal body weight being treated. Administration by the parenteral route(intravenously, intradermally, intraperitoneally, intramuscularly orsubcutaneously is preferred for a period of time of from 1 to 20 days.

While it is possible for the agents to be administered as the rawsubstances it is preferable, in view of their potency, to present themas a pharmaceutical formulation. The formulations, both veterinary andfor human use, of the present invention comprise the agent, togetherwith one or more acceptable carriers therefor and optionally othertherapeutic ingredients. The carrier(s) must be "acceptable" in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. Desirably, theformulations should not include oxidizing agents and other substanceswith which the agents are known to be incompatible. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing into association the agent with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the agent with the carrier(s) and then, if necessary,dividing the product into unit dosage's thereof.

Formulations suitable for parenteral administration convenientlycomprise sterile aqueous preparations of the agents which are preferablyisotonic with the blood of the recipient. Suitable such carriersolutions include phosphate buffered saline, saline, water, lactatedringers or dextrose (5% in water). Such formulations may be convenientlyprepared by admixing the agent with water to produce a solution orsuspension which is filled into a sterile container and sealed againstbacterial contamination. Preferably sterile materials are used underaseptic manufacturing conditions to avoid the need for terminalsterilization.

Such formulations may optionally contain one or more additionalingredients among which may be mentioned preservatives, such as methylhydroxybenzoate, chlorocresol, metacresol, phenol and benzalkoniumchloride. Such materials are of especial value when the formulations arepresented in multi-dose containers.

Buffers may also be included to provide a suitable pH value for theformulation and suitable materials include sodium phosphate and acetate.Sodium chloride or glycerin may be used to render a formulation isotonicwith the blood. If desired, the formulation may be filled into thecontainers under an inert atmosphere such as nitrogen or may contain anantioxidant, and are conveniently presented in unit dose or multidoseform, for example, in a sealed ampoule.

It will be appreciated that while the agents described herein form acidaddition salts and carboxy acid salts the biological activity thereofwill reside in the agent itself. These salts may be used in human and inveterinary medicine and presented as pharmaceutical formulations in themanner and in the amounts (calculated as the base) describedhereinabove, and it is then preferable that the acid moiety bepharmacologically and pharmaceutically acceptable to the recipient.Examples of such suitable acids include (a) mineral acids: hydrochloric,hydrobromic, phosphoric, metaphosphoric, and sulphuric acids; (b)organic acids: tartaric, acetic, citric, malic, lactic, fumaric,benzoic, glycollic, gluconic, gulonic, succinic and aryl-sulphonic, forexample, p-toluenesulphonic acids.

Surprisingly, the compounds of the invention have also demonstratedinsecticidal properties. The compounds have been found to beparticularly effective against mosquitoes.

EXAMPLE 8

Mosquito eggs (1,000) were hatched at 25° C. in a cultured mediaconsisting of well water (500 mL), baker's yeast (200 mg) and liverextract (300 mg). The eggs were maintained under these conditions for 4to 5 days. The larvae were next transferred to test tubes containing 3mL of culture media. Each test tube contained 10 mosquito larvae. Ineach experiment, 23 test tubes, each with 10 mosquitos in it, served ascontrols. The cidal activity of each of the polyamine analogues againstthe mosquito larvae was tested at, 1, 3, 10, and 30 ppm. Each compoundwas tested at each concentration in triplicate again in test tubescontaining 10 mosquito larvae in 3 mL of culture media, maintained at25° C. The control and test larvae were examined for insect death at 24and 48 hour intervals. Table 9 includes representative examples of thecidal activity of the polyamine analogues against mosquito larvae. Thedata is reported as the LD₅₀ values for each compound, i.e., theconcentration of polyamine required to kill 50% of the larvae.Furthermore, the data is reported at 48 and 96 hours.

                  TABLE 9                                                         ______________________________________                                        Compound          48 Hr. LD.sub.50                                                                        96 Hr. LD.sub.50                                  ______________________________________                                        N.sup.1,N.sup.4 -Diethyl spermine                                                               2 ppm     --                                                N.sup.1,N.sup.4 -Diethyl homospermine                                                           7 ppm     5 ppm                                             ______________________________________                                    

The insecticidal compounds of the invention may be dissolved ordispersed in any suitable carrier medium adapted for sprayinginsecticides, e.g., water or other aqueous media and sprayed on aninsect infested area or areas subject to potential infestation. Theconcentration of polyamines applied to an area would depend on thespecies of insect and its accessibility, however, solutions containingfrom 10 to 10,000 ppm per gallon broadcast over 100 ft².

We claim:
 1. A method of controlling the growth of mosquitos comprisingadministering to said mosquitos a mosquito insecticidally effectiveamount of a compound having one of the formulae: ##STR14##

    R.sub.1 --N.sup.1 H--(CH.sub.2).sub.3 --N.sup.2 H--(CH.sub.2).sub.3 --N.sup.3 H--(CH.sub.2).sub.4 --N.sup.4 H--(CH.sub.2).sub.3 --N.sup.5 H--(CH.sub.2).sub.3 --N.sup.6 H--R.sub.6                  (II); ##STR15## wherein R.sub.1 and R.sub.6 may be the same or different and are alkyl having from 1 to 12 carbon atoms or hydrocarbyl aralkyl having up to 12 carbon atoms;

R_(2-R) ₅ may be the same or different and are H, R₁ or R₆ ; R₇ is H,alkyl having from 1 to 12 carbon atoms, hydrocarbyl aryl or hydrocarbylaralkyl, each having up to 12 carbon atoms; m is an integer from 3 to 6,inclusive; n is an integer from 3 to 6, inclusive; or (IV) a saltthereof with an acid and a carrier therefor.